miRNAs的表达与原发性IgA肾病病情相关性初步研究

发布时间：2018-03-19 09:13

  本文选题：IgA肾病　切入点：miRNAs　出处：《郑州大学》2014年硕士论文　论文类型：学位论文

【摘要】：背景 IgA肾病是全世界最常见的原发性肾小球疾病，容易进展至终末期肾脏病(end stage renal disease, ESRD)。但是，IgA肾病的诊断和动态检测依赖肾组织活检，而肾活检为有创性的检查，且重复肾活检患者多难以接受。因而，探索IgA肾病非侵入性的诊断方法任重道远。miRNAs是一种参与转录后水平调控的非编码RNA，其表达异常可能导致人类多种疾病，包括IgA肾病，异常表达的miRNAs可以导致IgA肾病患者肾组织细胞结构的改变及类型的转化、免疫分子结构的改变、细胞信号传导的异常等，从而引起肾组织损伤。目前，众多的研究发现血浆及尿液中存在稳定表达的miRNAs，可抵制RNase的降解。因此，血浆或尿液中与IgA肾病病理形态学改变相关的miRNAs或许可以成为其新的诊断标志物。然而，miRNAs的表达水平与IgA肾病病情的关系尚无确切结论，血浆或尿液中miRNAs的表达水平是否能够真正取代肾活检仍值得探讨。本研究选取与IgA肾病发病和肾脏纤维化相关的几种miRNAs，通过对IgA肾病患者的肾组织、血浆及尿液miRNAs同时进行检测，初步研究肾组织、血浆及尿液miRNAs的表达与IgA肾病病情的相关性。 目的 通过检测miR-148b、 miR-194、 miR-200a及miR-382在原发性IgA肾病肾组织、血浆及尿液中的表达水平，初步探讨miRNAs的表达与该疾病病情的相关性，为血浆及尿液中的miRNAs在IgA肾病诊断中的应用提供参考，也为寻找IgA肾病非侵入性诊断标志物提供新思路。 方法 1.选取郑州大学第一附属医院肾脏内科2012年12月至2013年6月首次经肾组织活检及临床、实验室检查综合确诊为原发性IgA肾病患者34例，平均年龄(32.74±10.57)岁，男女比例1:1.1。该34例入组患者在肾活检当日清晨同时留取晨尿及空腹静脉血标本，其中7例患者留取肾穿刺组织标本。收集所有入组患者的临床及病理资料，并对其病理结果进行Hass分级，其中符合Hass分级I-II级患者10例，III级17例，IV-V级7例。对照组选自我院同期门诊年龄及性别匹配的健康体检者13例，每个入组对象同时留取晨尿及空腹静脉血标本，对照组肾组织标本选自同期在我院行肾肿瘤切除术的患者7例，取远离肿瘤的正常肾组织。 2.采用TRIzol LS Reagent（Invitrogen life technologies）抽提肾组织、血浆及尿液中的RNA，使用NanoDrop ND-1000(Nanodrop, Wilmington, Delaware,USA)测定RNA的浓度和纯度，应用实时荧光定量PCR(real-time PCR)技术定量检测肾组织、血浆及尿液中miR-148b、miR-194、miR-200a及miR-382的表达水平。选择RNU6B作为内参，数据采用2-△△Ct法进行分析。 3.比较（1）IgA肾病组和对照组间肾组织、血浆及尿液4种miRNAs的表达水平是否有差异；（2） IgA肾病患者血浆及尿液4种miRNAs的表达水平与Hass分级的关系；（3）IgA肾病患者血浆及尿液4种miRNAs的表达水平与临床指标的关系；（4）IgA肾病患者血浆及尿液4种miRNAs的表达水平与牛津病理分型的关系。正态分布数据两组间比较采用t检验，多组间比较采用One-way ANOVA，多个变量间的两两比较采用Bonferroni检验，将检验水准校正为0.017（0.05/3）。相关性检验应用Pearson相关（服从正态分布的资料）或Spearman秩相关（不服从正态分布的资料）。以P＜0.05表示差异有统计学意义。 结果 纳入IgA肾病组血浆及尿液标本各34例，肾穿刺组织标本7例；对照组血浆及尿液标本各13例，正常肾组织标本7例。 （1）IgA肾病组和对照组间肾组织、血浆及尿液miRNAs表达水平的比较：肾组织miR-148b、miR-194、miR-200a的表达水平下调（P＜0.05），miR-382的表达水平上调（P＜0.05）；血浆miR-148b、miR-194、miR-382的表达水平下调（P＜0.05），miR-200a的表达水平上调（P＜0.05）；尿液miR-148b、miR-382的表达水平上调（P＜0.05），miR-194、miR-200a的表达水平下调（P＜0.05）。 （2）不同Hass分级IgA肾病患者血浆miRNAs表达水平分析：miR-148b和miR-194的表达水平随IgA肾病Hass分级的升高而下降（P＜0.017）。Hass分级III级和IV-V级患者miR-382的表达水平低于I-II级（P＜0.017）。Hass分级I-II级、III级和IV-V级患者miR-200a的表达水平无显著性差异（P＞0.05）。 （3）不同Hass分级IgA肾病患者尿液miRNAs表达水平的分析：IgA肾病Hass分级IV-V级患者miR-148b的表达水平高于III级（P＜0.017）。Hass分级I-II级和III级患者miR-194的表达水平高于IV-V级（P＜0.017）。Hass分级I-II级、III级和IV-V级患者miR-200a、miR-382的表达水平无显著性差异（P＞0.05）。 （4）IgA肾病患者血浆及尿液miRNAs的表达水平与临床指标的相关性分析：IgA肾病患者尿液miR-148b的表达水平与血清尿酸、血清补体C4呈负相关性（P＜0.05），尿液miR-382的表达水平与尿红细胞计数呈正相关性（P＜0.05）。尿液miR-194、miR-200a及血浆4种miRNAs与血清肌酐、肾小球滤过率、血尿素氮、血尿酸、血胱抑素、血总蛋白、血白蛋白、血球蛋白、血清总胆固醇、血清三酰甘油、血清高密度脂蛋白、血清低密度脂蛋白、血红蛋白、24小时尿蛋白定量、尿红细胞计数，，血清补体C3、血清补体C4、血压均无显著相关性（P＞0.05）。 （5）不同牛津病理分型IgA肾病患者血浆及尿液miRNAs表达水平的比较：牛津病理分型T0型IgA肾病患者尿液miR-200a的表达水平高于T1-2型（P＜0.05），尿液miR-148b、miR-194、miR-382及血浆4种miRNAs的表达水平在E、S、T分型间无显著性差异（P＞0.05）。 结论 IgA肾病组肾组织、血浆及尿液miR-148b、miR-194、miR-200a、miR-382的表达水平与对照组相比均存在显著性差异，且尿液miRNAs的表达水平与肾组织一致性较好。血浆miR-148b、miR-194、miR-382及尿液miR-148b、miR-194、miR-200a的表达与IgA肾病病理损伤程度相关，尿液miR-148b、miR-382的表达与部分临床指标相关。提示miRNAs可能参与了IgA肾病的发生及进展，miRNAs是否能够成为IgA肾病无创性诊断标志物，有待进一步研究。
[Abstract]:background
IgA nephropathy is the world's most common primary glomerular disease, easy progression to end-stage renal disease (end stage renal disease, ESRD). However, the diagnosis and dynamic detection of IgA nephropathy on renal biopsy, and renal biopsy is an invasive examination, and repeated renal biopsy patients more difficult to accept. Therefore, to explore IgA nephropathy by non-invasive diagnostic methods to.MiRNAs is a non RNA encoding involved in post transcriptional regulation, its abnormal expression may lead to many diseases, including IgA nephropathy, abnormal expression of miRNAs can lead to IgA nephropathy patients with renal cell structural changes and type transformation, the structure of immune molecule change of cell signal conduction abnormalities, resulting in renal tissue damage. At present, many studies have found that the existence of stable expression of miRNAs in plasma and urine, can resist degradation of RNase. Therefore, plasma or urine Associated with the pathological change of IgA nephropathy miRNAs may become a new diagnostic marker. However, the relationship between the expression level of miRNAs in patients with IgA nephropathy is no definite conclusion, whether the expression level of miRNAs in plasma or urine can truly replace renal biopsy is still worthy of discussion. With the selection of several miRNAs related disease and renal fibrosis IgA nephropathy in this study, patients with IgA nephropathy, plasma and urinary miRNAs were detected at the same time, a preliminary study on the correlation between the expression of renal tissue, plasma and urine miRNAs in patients with IgA nephropathy.
objective
Through the detection of miR-148b, miR-194, miR-200a and miR-382 in primary renal tissue of IgA nephropathy, the expression level in plasma and urine, to study the correlation between the expression of miRNAs and severity of the disease, to provide reference for the application of plasma and urine miRNAs in the diagnosis of IgA nephropathy, also for non-invasive diagnosis of IgA nephropathy markers provide a new way of thinking.
Method
1. the First Affiliated Hospital of Zhengzhou University Department of nephrology in December 2012 to June 2013 for the first time by biopsy and clinical laboratory examination of renal tissue, comprehensive diagnosis of primary IgA nephropathy patients 34 cases, average age (32.74 + 10.57) years of age, the proportion of men and women 1:1.1. the 34 patients in the renal biopsy at the same time, day morning urine and fasting blood samples were collected, including 7 cases of patients with renal biopsies were collected. All the patients enrolled in the clinical and pathological data, and the pathological results were Hass grade, with 10 cases of Hass grade I-II patients, 17 cases of grade III, IV-V grade 7 cases. The control group self selected hospital healthy outpatient age and sex matched 13 cases, each group and urine and fasting venous blood samples of normal renal specimens were selected from the same period in our hospital underwent renal tumor resection in 7 cases, from normal renal tumor Organization.
2. using TRIzol LS Reagent (Invitrogen life technologies) in renal tissue from plasma and urine in RNA, using NanoDrop ND-1000 (Nanodrop, Wilmington, Delaware, USA) for determination of the concentration and purity of RNA, the application of real-time fluorescence quantitative PCR (real-time PCR) in renal tissue of quantitative detection technique, miR-148b, plasma and urine miR-194 expression the level of miR-200a and miR-382. RNU6B is selected as the reference data, using 2- Delta Ct method were analyzed.
3. (1) IgA nephropathy group and control group in renal tissues, the expression level of plasma and urine of 4 kinds of miRNAs whether there are differences; (2) the relationship between the expression level and Hass grade in the plasma and urine of IgA nephropathy in 4 miRNAs; (3) the relationship between the plasma and urine of IgA nephropathy 4 miRNAs. Level and clinical index; (4) the relationship between the expression level of the plasma and urine of IgA nephropathy in 4 miRNAs Oxford and pathologic type. The normal distribution data of the two groups were compared using t test and multiple comparisons with One-way ANOVA, a variable between 22 compared with Bonferroni test, the test level the correction was 0.017 (0.05/3). The correlation test using Pearson (normal distribution data) or Spearman rank correlation (not subject to normal distribution data). P < 0.05 said the difference was statistically significant.
Result
The plasma and urine specimens of IgA nephrotic group were included in 34 cases, renal biopsy specimens in 7 cases, and in the control group, 13 cases of plasma and urine specimens, and 7 normal renal tissue specimens.
(1) IgA nephropathy group and the control group of renal tissue, plasma and urine miRNAs levels: miR-194, miR-148b in renal tissue, the expression level of miR-200a decreased (P < 0.05), the expression level of miR-382 (P < 0.05); plasma miR-148b, miR-194, the expression level of miR-382 decreased (P < 0.05), the expression level of miR-200a (P < 0.05); urine miR-148b, the expression level of miR-382 (P < 0.05), miR-194, the expression level of miR-200a decreased (P < 0.05).
(2) the level of analysis of classification of IgA nephropathy patients plasma miRNAs with different Hass expression: the expression of miR-148b and miR-194 decreased with the increase of IgA nephropathy Hass grading (P < 0.017) the expression level of.Hass III grade and IV-V grade in patients with miR-382 less than I-II (P < 0.017).Hass grade I-II, no significant difference the expression level of III and IV-V in patients with miR-200a (P > 0.05).
(3) the level of classification analysis of urine miRNAs in patients with IgA nephropathy with different Hass expression: the expression level of IgA nephropathy Hass IV-V grade miR-148b patients was higher than that of grade III (P < 0.017) expression of.Hass class I-II and III level in miR-194 patients was higher than that of grade IV-V (P < 0.017).Hass grade I-II, grade III and IV-V patients with grade miR-200a, no significant difference in the expression level of miR-382 (P > 0.05).
(4) analysis of the correlation between the expression level of IgA in plasma and urine of miRNAs nephropathy patients with clinical indicators: the expression level of serum uric acid and urine miR-148b in patients with IgA nephropathy, there was a negative correlation between serum C4 (P < 0.05), urine miR-382 expression level and urine red blood cell count showed a positive correlation (P < 0.05) urine. MiR-194, miR-200a and miRNAs 4 plasma and serum creatinine, glomerular filtration rate, blood urea nitrogen, uric acid, blood cystatin, serum total protein, albumin, globulin, serum total cholesterol, serum three triglycerides, serum high density lipoprotein, serum low density lipoprotein cholesterol, hemoglobin, urine protein in 24 hours, urine red blood cell count, serum C3, serum C4, blood pressure had no significant correlation (P > 0.05).
Oxford (5) of different pathological type of IgA nephropathy patients plasma and urine miRNAs levels: the expression level of Oxford pathological urine miR-200a in patients with type T0 type of IgA nephropathy is higher than that of the T1-2 (P < 0.05), urine miR-148b, miR-194, expression level of miR-382 and plasma water of 4 miRNAs in E, S, T no significant difference (P > 0.05).
conclusion
IgA nephropathy group renal tissue, plasma and urine miR-148b, miR-194, miR-200a, miR-382 expression level compared with the control group there were significant differences, and the expression level of miRNAs in urine and renal tissue consistency. Plasma miR-148b, miR-194, miR-382 and miR-148b in urine, miR-194, expression of miR-200a, IgA and renal pathological injury the degree of urine miR-148b, miR-382 expression correlated with some clinical parameters. It suggests that miRNAs may participate in the occurrence and progression of IgA nephropathy, miRNAs nephropathy can be IgA noninvasive diagnostic markers, require further research.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692.3

【参考文献】

相关期刊论文 前8条

1 付莎;徐安平;;IgA肾病尿液生物标记物的研究进展[J];国际内科学杂志;2009年10期

2 文璐;文建国;李真珍;刘章锁;;MicroRNA在IgA肾病诊断中的应用[J];河南医学研究;2013年04期

3 刘畅;饶向荣;;MicroRNA在肾脏疾病中的研究进展[J];中国中西医结合肾病杂志;2011年02期

4 俞焙秦;刘炳亚;;miRNA的生物学特性和功能[J];上海交通大学学报(医学版);2007年05期

5 俞雨生;王金泉;;IgA肾病临床及其发病机制的新观点[J];肾脏病与透析肾移植杂志;2006年01期

6 张昌明;张婉芬;刘春蓓;曾科;刘志红;;循环microRNAs与局灶节段性肾小球硬化患者疾病活动的关系[J];肾脏病与透析肾移植杂志;2013年04期

7 巴建明,朱开元,李慧云,吴增常;转化生长因子-β及其受体在人类IgA肾病的表达[J];上海医学;2004年05期

8 刘玉梅;王筱霞;汪年松;;microRNA在肾脏病中的研究进展[J];中华临床医师杂志(电子版);2012年10期

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